The present invention relates generally to p53 and its use in treatment of conditions associated with inappropriate levels of p53 tumor suppressor activity.
Wild-type p53 is a sequence-specific transcription factor that induces cell cycle arrest or programmed cell death in response to DNA damage [W. Maltzman and L. Czyzyk, Mol. Cell. Biol., 4:1689-1694 (1984); M. B. Kastan et al., Cancer Res., 51: 6304-6311 (1991); S. W. Lowe, et. al., Cell, 74: 957-967 (1993); S. W. Lowe, et. al., Nature, 362: 847-849 (1993); A. R. Clarke, et. al., Nature. 362: 849-852 (1993); A. D. Leonardo, et. al., Genes Dev., 8: 2540-2551 (1994)]. The N-terminus of p53 contains a transactivation domain [S. Fields and S. K. Jang, Science 249: 1046-1049 (1990); T. Unger et al., EMBO J. 11: 1383-1390 (1992)], the central region a sequence-specific DNA binding domain [J. Bargonetti, et. al., Genes Dev., 7: 2565-2574 (1993); N. P. Pavletich, et. al., Genes Dev, 7: 2556-2564 (1993); Y. Wang et. al., Genes Dev, 7: 2575-2586 (1993); T. D. Halazonetis and A. N. Kandil, et. al., EMBO J. 12: 5057-5064 (1993)], and the C-terminus a tetramerization domain [J. Milner, et. al., Mol. Cell. Biol. 11: 12-19 (1991); H. W. Stxc3xcrzbecher, et. al., Oncogene, 7: 1513-1523 (1992); H. Sakamoto, et. al., Proc. Natl. Acad. Sci. U.S.A. 91: 8974 (1994); P. Wang, et. al, Mol. Cell. Biol. 14: 5182-5191 (1994)]. The tetramerization domain mediates homo-oligomerization, which is required for high affinity sequence-specific DNA binding activity and tumor suppressor function [Pietenpol et. al., Proc. Natl. Acad. Sci. USA, 91:1998-2002 (1994)].
In about half of all human tumors, the sequence-specific DNA binding domain of p53 is inactivated by point mutations [C. C. Harris, Science, 262: 1980-1981 (1993); S. Friend, Science, 265: 334-335 (1994); J. Bargonetti, et. al., Cell, 65: 1083-1091 (1991); S. E. Kern et. al., Science 252: 1708-1711 (1991)]. The tumor-derived p53 mutants fail to suppress tumor growth [L. Diller, et. al., Mol. Cell. Biol., 10: 5772-5781 (1990); S. J. Baker, et. al., Science, 249: 912-915 (1990); D. Eliyahu, et. al, Proc. Natl. Acad. Sci., USA, 86: 8763-8767 (1989); W. E. Mercer, et. al., Proc. Natl. Acad. Sci. USA, 87: 6166-6170 (1990); C. A. Finlay, et. al., Cell, 57: 1083-1093 (1989)] and also transdominantly inhibit wild-type p53 [J. Martinez, et. al., Genes Dev., 5: 151-159 (1991); J. Bargonetti, et. al, Genes Dev., 6: 1886-1898, (1992); S. E. Kern, et. al., Science, 256: 827-830 (1992); J. Miner and E. A. Medcalf, Cell, 65: 765-774 (1991); D. Eliyahu, et. al, Nature 312: 646-649 (1984); L. F. Parada, et. al., Nature 312: 649-651 (1984); J. R. Jenkins, et. al., Nature 312: 651-654 (1984)]. There is significant evidence that transdominant inhibition of wild-type p53 is mediated by sequestration of wild-type p53 into inactive mutant/wild-type heterotetramers. The ability of tumor-derived p53 mutants to transdominantly inhibit wild-type p53 requires an intact tetramerization domain. The isolated p53 tetramerization domain also inhibits wild-type p53 function [T. Unger, et. al., Mol. Cell. Biol., 13: 5186-5194 (1993); E. Shaulian, et. al., Mol. Cell. Biol., 12: 5581-5592 (1992); M. Reed, et. al., Gene Expression 3: 95-107 (1993)]. Chimeric p53 proteins that contain a heterologous tetramerization domain, instead of the native p53 tetramerization domain, are not transdominantly inhibited by tumor-derived p53 mutants.
Induction of wild-type p53 function in tumor cells leads to growth arrest or apoptosis (L. Diller, cited above; S. J. Baker, cited above; W. E. Mercer, cited above; E. Yonish-Rouach, et. al., Nature, 352: 345-347 (1991); P. Shaw, et. al., Proc. Natl. Acad. Sci. USA, 89: 4495-4499 (1992)]. Thus, introduction of wild-type p53 into tumor cells could in principle be utilized for therapy [T. Fujiwara, et. al., Cancer Res., 53: 4129-4133 (1993); T. Fujiwara, et. al., Cancer Res., 54: 2287-2291 (1994); T. Liu, et. al., Cancer Res., 54: 3662-3667 (1994). One obstacle to the effectiveness of such therapy, however, is that about half of all human tumors express dominant negative p53 mutants [Harris et al, cited above, S. Friend et al, cited above, J. Bargonetti et al, cited above; S. E. Kern et. al., cited above.].
The design and analysis of a p53 chimeric protein in which the native p53 oligomerization domain is substituted with a modified leucine zipper that assembles as a tetramer has been reported [See, e.g., U. S. Pat. No. 5,573,925]. This engineered p53 protein is not transdominantly inhibited by tumor-derived p53 mutants, but is not as active as wild-type p53.
What are needed are constructs and methods for providing p53 tumor suppressor activity, particularly in the presence of insufficient activity due to the presence of tumor-derived p53 mutants or other factors.
In a first aspect, the present invention provides a modified p53 protein containing a p53 DNA binding domain and a modified p53 tetramerization domain that does not hetero-oligomerize with the native p53 tetramerization domain. The modified tetramerization contains the sequence of amino acid residues 325 to 355 of a human p53 or a homologous p53 sequence which has been modified at one or more of the following residues (with reference to the numbering scheme of the human p53 in SEQ ID NO: 2): Leu330 substituted with Phe; Met340 substituted with Phe; Ala347 substituted with Ile; Leu348 substituted with Met; Ala353 substituted with Leu; Gln354 substituted with Leu; and/or Ala355 substituted with Asp.
In another aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier; and a modified p53 protein of the invention.
In still another aspect, the present invention provides a nucleic acid molecule encoding a modified p53 protein of the invention. Optionally, this molecule may further contain regulatory sequences which direct expression of said modified tetramerization domain or modified p53 protein.
In yet another aspect, the present invention provides a vector containing a nucleic acid sequence of the invention and sequences which direct expression thereof
In still a further aspect, the present invention provides a pharmaceutical composition comprising a nucleic acid sequence of the invention and a pharmaceutically acceptable carrier.
In yet a further aspect, the present invention provides a method of treating a condition associated with abnormal p53 activity comprising the step of administering a pharmaceutical composition as described above.
Other aspects and advantages of the present invention are described further in the detailed description of the preferred embodiments thereof
The present invention provides modified p53 proteins containing modified tetramerization domains having altered oligomerization specificity. The modified p53 proteins of the invention form homo-tetramers and have tumor suppressor function, but do not hetero-oligomerize with tumor-derived p53 mutants and resist transdominant inhibition.
The modified tetramerization domains of the invention contain at least one of the following substitutions within the hydrophobic core of the p53 tetramerization domain, which is located in the region of about amino acids 325 to about 355, with reference to human p53: Leu330 substituted with Phe; Met340 substituted with Phe; Ala347 substituted with Ile; Leu348 substituted with Met; Ala353 substituted with Leu; Gln354 substituted with Leu and/or Ala355 substituted with Asp. In a currently preferred embodiment, a modified p53 protein construct of the invention contains a modified human p53 tetramerization domain, with all seven of the amino acid substitutions identified herein.
The present inventors have found that the modified p53 tetramerization domains, when engineered into modified p53 protein constructs block the interaction of wild-type p53 with tumor-derived p53 mutants. The modified p53 tetramerization domains of the invention contain amino acid substitutions within the p53 tetramerization domain that change oligomerization specificity. The p53 tetramerization domains modified in this way still forms tetramers, but do not hetero-oligomerize with the unmodified (native) domain. The inventors have found that, unlike the prior art constructs which utilized leucine zippers which cause oligomerization specificity to be determined primarily by charged residues, conservative hydrophobic amino acid substitutions change the oligomerization specificity of the p53 tetramerization domain.
All references to human p53 residue numbers herein refer to the numbering scheme provided by Zakut-Houri et al, (1985) EMBO J., 4: 1251-1255 (1985) [GenBank Code Hsp53], which is incorporated by reference, and reproduced in SEQ ID NOS: 1 and 2However, one of skill in the art will readily understand that the sequences referenced herein may be obtained by a variety of known techniques including, e.g., chemical synthesis. Additionally, where reference is made to the human p53 residue numbers, one of skill in the art will readily understand that corresponding sequences from homologous p53 sequences may be readily substituted. For example, the corresponding sequences (e.g., to amino acid residues 325 to 355 of human p53, SEQ ID NO: 2) may be readily determined by reference to the alignment of the human sequences to a variety of non-human p53 sequences provided in Soussi et al, Oncogene, 5: 945-952 (1990), cited above. The alignment of additional p53 sequences homologous to human p53 may be obtained by commercially available computer programs, such as BESTFIT of the University of Wisconsin GCG package. The homolog of p53 which can be used to substitute for the human p53 sequences can be readily selected by one of skill in the art.
Desirably, these modified tetramerization domains are contained within a protein construct which further contains a p53 DNA binding domain.
I. P53 Protein Constructs
The present invention provides a modified p53 protein construct which contains a p53 DNA binding domain and a modified tetramerization domain, as described herein. These p53 protein constructs may contain sequences from a single source or from multiple sources, e.g., these constructs may contain sequences which were chemically synthesized, sequences which were derived from a human p53, sequences which were derived from a non-human p53, or combinations thereof.
As defined herein, chimeric p53 protein constructs of the invention include proteins containing an N-terminal portion of p53 containing at least a DNA binding domain fused, optionally via a suitable linker, to the modified p53 tetramerization domain described herein. When utilized, linkers may be readily selected from among those known to those of skill in the art. While these linkers may be chemical compounds or other inorganic constructs, they are more desirably amino acids or peptides. Regardless of the type of linker used, the linkers do not interfere with the function of the p53 chimeric protein. See, U.S. Pat. No. 5,573,925 for examples of suitable linkers.
The ability of p53 to bind DNA in a sequence-specific manner maps to about amino acid 93 to about amino acid 293 of human p53, SEQ ID NO: 2 [Halazonetis and Kandil, EMBO J., 12: 5057-5064 (1993); Pavletich et al, Genes Dev., 7: 2556-2564 (1993); Wang et al, Genes Dev., 7: 2575-2586 (1993)]. Thus, this region is a particularly suitable DNA binding domain. However, modifications to this region which do not substantially reduce the p53 DNA binding ability and/or specificity may be incorporated into the p53 protein constructs of the invention. Such modifications include naturally occurring or engineered mutants. For example, suitable mutants include, without limitation, p53 having glutamine at residue 248 (p53Q248), p53 having histidine at residue 273 (p53H273), and p53 having cysteine at residue 273 (p53C273). Other p53 mutants include those having a mutation of the native Thr284 to Arg or Lys, as described in WO 97/10843.
The p53 constructs of the invention may further contain native p53 N-terminal (i.e., residues about 1 to about 93 of human p53, SEQ ID NO: 2) and C-terminal residues (i.e., residues about 256 to about 393 of human p53, SEQ ID NO: 2). Alternatively, the p53 constructs of the invention may contain deletions or modification in either of the N-terminus or C-terminus, as desired. One particularly desirable N-terminal deletion involves substitution of the p53 transaction domain (e.g., about aa 3 to about aa 80 of human p53, SEQ ID NO: 2) with the transactivation domain derived from another protein (e.g., the herpes simplex virus protein VP16 domain, aa residues 402-479 of VP16, described in Pellett et al, Proc. Natl. Acad. Sci. USA, 82:5870-5874 (1985); GenBank Code He11cg]. One particularly desirable C-terminal deletion involves truncation of about amino acid 364 to about amino acid 393 of human p53, SEQ ID NO: 2However, suitable deletions include truncation following amino acid residue 355, and deletions internal to the region corresponding to residues 356-393 of human p53, SEQ ID NO: 2.
A currently preferred modified p53 protein construct of the invention, which contains full-length p53 modified to contain a p53 tetramerization domain of the invention, is termed herein p53IND7 [SEQ ID NO: 3]. P53IND7 has very similar properties to the wild-type tetramerization domain. Both domains assemble as tetramers and support the sequence-specific DNA binding, transactivation and tumor suppressor functions of p53Furthermore, p53IND7 is anticipated to have a three-dimensional structure that is very similar to the structure of the wild-type domain. In fact, because six of the seven substitutions involve buried residues, the surfaces of the p53wt [SEQ ID NO: 2] and p53IND7 [SEQ ID NO: 3] domains may be remarkably similar.
The high functional and structural similarity between the p53IND7 [SEQ ID NO: 3] and p53wt [SEQ ID NO: 2] domains, suggests that p53IND7 can be used to study p53 function in cells containing high levels of endogenous wild-type or mutant p53More significantly, p53IND7 could be used in a gene therapy approach for treatment of human cancer [T. Fujiwara et al., Cancer Res., 53:4129-4133 (1993); T. Figiwara et al, Cancer Res., 54:2287-2291 (1994); T. J. Liu et al, Cancer Res., 54:3662-3667 (1994)]. A p53 protein with altered oligomerization specificity would be clearly superior to wild-type p53 for such therapy, since about half of all human tumors express p53 mutants that can inhibit wild-type p53 activity [C. C. Harris, cited above; S. Friend, cited above].
The modifications described herein may be engineered using known techniques, e.g., by chemical synthesis techniques or by mutating the nucleic acid sequences encoding the amino acid which is to be altered using conventional site-directed mutagenesis techniques [R. Higuchi et al, in M. A. Innis et al, (eds.), PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, pp. 177-183 (1990)].
II. Nucleic Acid Sequences Encoding Modified p53 Proteins of the Invention
The present invention further provides nucleic acid sequences encoding the modified p53 proteins and tetramerization domains of this invention. In addition to the coding strand, the nucleic acid sequences of the invention include the complementary DNA sequence representing the non-coding strand, the messenger RNA sequence, the corresponding cDNA sequence and the RNA sequence complementary to the messenger RNA sequence. Variants of these nucleic acids of the invention include variations due to the degeneracy of the genetic code and are encompassed by this invention. Such variants may be readily identified and/or constructed by one of skill in the art. In certain cases specific codon usage may be employed to optimize expression. The above nucleotide sequences can be included within larger DNA or RNA fragments, or may be interrupted by introns.
A. Expression Vectors
In another embodiment, the nucleic acids encoding the proteins of the invention are present in the context of vectors suitable for amplification in prokaryotic or eukaryotic cells. Many such vectors are known and many of these are commercially available. For example plasmids with bacterial or yeast replication origins allow amplification in bacteria or yeast, respectively. Such vectors allow the production of large quantities of nucleic acids encoding the proteins of the invention, which nucleic acids can be used for gene therapy or for expression of the modified p53 proteins of the invention.
In yet another embodiment the nucleic acids encoding the proteins of the invention are present in the context of vectors suitable for expression in cell-free extracts or lysates or in prokaryotic or eukaryotic cells. Many such vectors are known [Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley and Sons, N.Y. (1997)] and many of these are commercially available. For example, the vector pGEM4 (Promega, Madison, Wis.) is suitable for expression of the proteins in cell-free lysates, while the vector pSV2 [ATCC] is suitable for expression in mammalian cells. Such vectors allow the production of the proteins of the invention in vitro for analysis of their functional properties or for delivery to patients either directly or indirectly (i.e., ex vivo).
B. Gene Therapy Vectors
The nucleic acid sequences of the invention may be inserted into a vector capable of targeting and infecting a desired cell, either in vivo or ex vivo for gene therapy, and causing the encoded modified protein of this invention to be expressed by that cell. Many such viral vectors are useful for this purpose, e.g., adenoviruses, retroviruses and adeno-associated viruses (AAV) [Schreiber et al., Biotechniques, 14: 818-823 (1993); Davidson et al., Nature Genetics, 3: 219-223 (1993); Roessler et al., J. Clin. Invest., 92: 1085-1092 (1993); Smythe et al., Ann. Thorac. Surg., 57: 1395-1401 (1994); Kaplitt et al, Nature Genetics, 8: 148-154 (1994)]. There has already been success using viral vectors driving expression of wild-type p53 [Fujiwara et al., Cancer Res., 53: 4129-4133 (1993); Fujiwara et al., Cancer Res., 54: 2287-2291 (1994); Friedmann, Cancer, 70(6 Suppl): 1810-1817 (1992); Fujiwara et al., Curr. Opin. Oncol., 6: 96-105 (1994b); Roth et al., Nature Medicine, 2:985-991 (1996)].
For use in gene therapy, these viral vectors containing nucleic acid sequences encoding a protein of the invention, e.g., a modified p53 protein, are prepared by one of skill in the art with resort to conventional techniques (see references mentioned above). For example, a recombinant viral vector, e.g. an adenovirus, of the present invention comprises DNA of at least that portion of the viral genome which is capable of infecting the target cells operatively linked to the nucleic acid sequences of the invention. By xe2x80x9cinfectionxe2x80x9d is generally meant the process by which a virus transfers genetic material to its host or target cell. Preferably, the virus used in the construction of a vector of the invention is rendered replication-defective to remove the effects of viral replication on the target cells. In such cases, the replication-defective viral genome can be packaged by a helper virus in association with conventional techniques.
Briefly, the vector(s) containing the nucleic acids encoding an altered protein of the invention is suspended in a pharmaceutically acceptable carrier, such as saline, and administered parenterally (or by other suitable means) in sufficient amounts to infect the desired cells and provide sufficient levels of modified protein to achieve the desired therapeutic or prophylactic effect, e.g., sufficient p53 activity to arrest abnormal cellular proliferation. Other pharmaceutically acceptable carriers are well known to those of skill in the art. A suitable amount of the vector containing the chimeric nucleic acid sequences is between about 106 to 109 infectious particles per mL carrier. The delivery of the vector may be repeated as needed to sustain satisfactory levels of biological activity. For example, where modified p53 is administered, activity may be determined by monitoring clinical symptoms.
As desired, this therapy may be combined with other therapies for the disease or condition being treated. For example, therapy involving the administration of a vector capable of expressing a modified p53 protein of the invention is well suited for use in conjunction with conventional cancer therapies, including surgery, radiation and chemotherapy.
Nucleic acid sequences driving expression of a protein of the invention may also be introduced by xe2x80x9ccarriersxe2x80x9d other than viral vectors, such as liposomes, nucleic acid-coated gold beads or can simply be injected in situ [Fujiwara et al (1994b), cited above; Fynan et al., Proc. Natl. Acad. Sci. USA, 90: 11478-11482 (1993); Cohen, Science, 259: 1691-1692 (1993); Wolffet a., Biotechnigues, II: 474-485 (1991)].
III. Pharmaceutical Compositions
The altered proteins and nucleic acid sequences of this invention may also be formulated into pharmaceutical compositions and administered using a therapeutic regimen compatible with the particular formulation. When administered in the form of nucleic acid sequences, the composition may contain xe2x80x9cnakedxe2x80x9d DNA, or a plasmid containing the nucleic acid sequences which may contain the regulatory elements necessary to drive expression of the modified p53 proteins of the invention. As used herein, the term xe2x80x9cnaked DNAxe2x80x9d means substantially pure DNA which is not associated with a protein, lipid, carbohydrate or contained within a cell or an artificial delivery system such as a liposome.
Pharmaceutical compositions within the scope of the present invention include compositions containing a modified protein of the invention (or a nucleic acid sequence encoding a modified protein) in an effective amount to have the desired physiological effect, e.g. to arrest the growth of cancer cells without causing unacceptable toxicity for the patient.
Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form, e.g. saline. Alternatively, suspensions of the active compounds may be administered in suitable conventional lipophilic carriers or in liposomes.
The compositions may be supplemented by active pharmaceutical ingredients, where desired. Optional antibacterial, antiseptic, and antioxidant agents in the compositions can perform their ordinary functions. The pharmaceutical compositions of the invention may further contain any of a number of suitable viscosity enhancers, stabilizers, excipients and auxiliaries which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Preferably, these preparations, as well as those preparations discussed below, are designed for parenteral administration. However, compositions designed for oral or rectal administration are also considered to fall within the scope of the present invention.
Those of skill in the pharmaceutical art should be able to derive suitable dosages and schedules of administration. As used herein, the terms xe2x80x9csuitable amountxe2x80x9d or xe2x80x9ceffective amountxe2x80x9d means an amount which is effective to treat the conditions referred to below. A preferred dose of a pharmaceutical composition containing a protein of this invention is generally effective above about 0.1 mg modified protein per kg of body weight (mg/kg), and preferably from about 1 mg/kg to about 100 mg/kg. These doses may be administered with a frequency necessary to achieve and maintain satisfactory activity levels. Although a preferred range has been described above, determination of the effective amounts for treatment of each type of tumor or other condition may be determined by those of skill in the art.
Dosage units of such pharmaceutical compositions containing the proteins of this invention preferably contain about 1 mg to 5 g of the protein.
IV. Therapeutic Indications
The nucleic acids encoding modified p53 proteins and the modified p53 proteins themselves can be introduced into human patients for therapeutic benefits in conditions characterized by insufficient wild-type p53 activity. Such conditions have been described in the art. See, e.g., PCT/US95/15353 (Jun. 6, 1996). For example, the pharmaceutical compositions of the invention, including the gene therapy vectors, may be employed to induce the cellular defense to DNA damaging agents such as sunlight UV irradiation, as well as radiation and chemotherapeutics used for cancer treatment. The therapeutic indications include inducing apoptosis of specific cells, such as proliferating lymphocytes, the prevention of transplant rejection, and the treatment of autoimmune diseases, e.g., systemic lupus erythrematosis, rheumatoid arthritis and the like.
The pharmaceutical compositions of this invention may also be employed to restore p53 function in tumor cells and to suppress cell proliferation in diseases other than cancers, which are characterized by aberrant cell proliferation. Among such diseases are included psoriasis, atherosclerosis and arterial restenosis. A variety of other suitable indications which will be readily apparent to one of skill in the art.
V. Antibodies
The modified p53 tetramerization domains and modified p53 protein constructs of the invention are useful for generating antibodies, which may be used as diagnostic reagents, for example, to monitor the presence of modified protein or modified tetramerization domain.
Specific antisera may be generated using known techniques. See, Sambrook, cited above, Chapter 18, generally, incorporated by reference. Similarly, antibodies of the invention, both polyclonal and monoclonal, may be produced by conventional methods, including the Kohler and Milstein hybridoma technique, recombinant techniques, such as described by Huse et al, Science, 246:1275-1281 (1988), or any other techniques known to the art.
The invention further encompasses functional fragments of the antibodies of the invention, including, Fab, Fv, and F(abxe2x80x2)2 fragments, the binding site of the antibodies, and the complementarity determining regions (CDRs). Optionally, the binding site and/or CDRs may be contained in a synthetic molecule which provides antibody framework regions. Further, these functional fragments may be used in the production of recombinant antibodies, including bifunctional antibodies, chimeric antibodies, and humanized antibodies, which preferably retain the antigen binding specificity of the antibodies of the invention. Such recombinant antibodies may be constructed and produced according to known techniques [see, e.g., S. D. Gillies et al, J. Immunol. Meth., 125:191-202 (1989); and G. E. Mark and E. A. Pladlan, xe2x80x9cHumanization of Monoclonal Antibodiesxe2x80x9d, The Handbook of Experimental Pharmacology, Vol. 113, Chapter 4, pp. 105-133, Springer-Verlag (June, 1994)]. These functional fragments and recombinant antibodies may be used for a variety of purposes, including any of those described herein for the antibodies of the invention.
In general, polyclonal antisera, monoclonal antibodies and other antibodies which bind to an altered protein as antigen are useful as research tools, as diagnostic reagents, as therapeutic agents, and for producing other antibodies (as described above) which are similarly useful.
VI. Diagnostic Reagents
The modified p53 proteins of the invention may be used as diagnostic reagents. These reagents may optionally be labeled using diagnostic labels, such as radioactive labels, colorimetric enzyme label systems and the like conventionally used in diagnostic or therapeutic methods. Alternatively, thexe2x80x94or C-terminus of a modified p53 protein of the invention may be tagged with a detectable label which can be recognized by a specific antisera. For example, the reagents derived from p53 may be used in diagnosis of a variety of conditions associated with p53 and/or aberrant cell proliferation, including autoimmune diseases, e.g., systemic lupus erythrematosis, rheumatoid arthritis and the like, cancers, psoriasis, atherosclerosis and arterial restenosis. For example, in tissue biopsies, the presence of p53 could be directly verified by RT-PCR or immunostaining. Reagents produced from other altered proteins of the invention, e.g., antibodies and the like, may similarly be utilized as diagnostic reagents. The selection of the appropriate assay format and label system is within the skill of the art and may readily be chosen without requiring additional explanation by resort to the wealth of art in the diagnostic area.
These examples illustrate the method of the invention as performed in connection with p53 and the preparation of modified p53 proteins of the invention. These examples are illustrative only and do not limit the scope of the invention.